A number of medical procedures pertaining to diagnosis and therapy require the administration to the patient of special chemicals in order to enhance the quality or degree of the anticipated result of the procedure. Representative examples of general classes of such chemicals include contrast-enhancing agents for Magnetic Resonance Imaging (MRI), fluor-tagged agents for fluorescent labeling of specific cells or organs, electron-dense agents for x-ray procedures, and radionuclide-labeled agents used to deliver radioactive isotopes to particular deep-seated tumors, and drug-labeled agents used to deliver multiple drug therapeutic molecules to the target site. Compounds administered for such purposes comprise molecules generally having one or more distinctive chemical groups or functional moieties attached thereto that give the molecule its desired pharmacologic utility.
As used herein, a pharmacologically active group or molecule is a group or molecule, respectively, having a chemical structure providing a desired diagnostic or therapeutic effect in the body of a human or other warm-blooded animal. For example, stable organic nitroxides are pharmacologically active because they serve as spin relaxers useful for contrast enhancement in the MRI diagnostic technique.
Many of the chemical compounds currently used as pharmacologic agents are toxic to the patient, especially in high dosages. In many cases, optimal results can only be obtained when the patient receives more of the agent than can be physiologically tolerated. In other words, the therapist is often faced with treading the fine line between obtaining the best possible pharmacologic result and either seriously intoxicating or killing the patient.
Other such compounds in current use, such as certain MRI contrast-enhancing agents, are so short-lived in a physiological environment that massive dosages need to be administered to obtain even a slight effect. For example, stable nitroxide free radicals are rapidly reduced when injected into the body. Nitroxide free radicals are metabolically converted in vivo to diamagnetic forms with consequent loss of their ability to provide contrast for MRI. In the past, "reduction" problems have been handled by injecting large amounts of simple nitroxide compounds, such as those containing a single nitroxide radical, into the patient with the intent of "swamping" the reduction reaction. Unfortunately, such large dosages of nitroxides are both toxic and cause osmotic disequilibria in the body.
In other instances, large dosages of pharmacologic chemicals are necessary because there are few practical ways to direct a particular compound to the desired target in the body where the compound can be concentrated for optimal effect. Without some way to render the compound "targeting," it is rapidly diluted in body circulatory liquid. To counteract this problem, massive dosages are sometimes administered in order to obtain a minimal concentration at the site of interest. Unfortunately, such large dosages can be toxic to the patient or otherwise cause serious physiochemical imbalances.
One way to concentrate diagnostic or therapeutic molecules at a given locus in the body is to attach the molecules to antibodies specific for (i.e., seroreactive with) the target cells of interest. To effect such attachment of a molecule, a bifunctional form thereof is first synthesized, comprising the pharmacologically active group on one end of the molecule, a protein-reactive-group on the other end, and a linker group serving as a structural bridge joining the ends of the molecule together. The protein-reactive group on the bifunctional molecule reacts with one or more specific moieties on the antibodies, yielding a population of conjugated antibodies ("decorated" or "tagged") having one or more of the desired diagnostic or therapeutic molecules chemically bonded thereto. One example of a protein-reactive group is the isothiocyanate moiety (--N.dbd.C.dbd.S). Isothiocyanate is reactive with the .epsilon.-amino group of lysine, a common amino acid in proteins, yielding a thiourea (--NHSNH--) linkage between the lysine and the linker, and the linker terminating with the pharmacologically active group. When the conjugated antibodies are administered to a patient, they carry the attached diagnostic or therapeutic agent to the target cells to which the antibodies become attached.
Although the conjugated antibody concept has been successful in some instances, it has several drawbacks when utilized with many existing pharmacologic agents that have only one active group per molecule. First, as reviewed above, some such agents, such as nitroxides for MRI, are short-lived in the body. Merely conjugating them to target-specific antibodies would help concentrate them at the desired target, but would not slow the rate at which the nitroxides are reduced in vivo. Attaching larger numbers of bifunctional nitroxides to the antibodies would seem to increase both the effective concentration of active nitroxide at the target and the time before the effective concentration drops below the minimal required level. Unfortunately, however, attaching too many conjugates to an antibody may destroy the antibody's immunological activity, i.e., its ability to attach to the target. If it were possible to attach fewer nitroxide conjugates to the antibody, where each conjugate has more than the single nitroxide group found in existing MRI contrast-enhancing agents, one would obtain antibodies that retain their ability to bind to the target while having a sufficient number of attached nitroxides to achieve the desired contrast of the target in a magnetic resonance image.
Therefore, the stated drawbacks of many existing diagnostic and therapeutic agents are due to their having only one or a very few diagnostically or therapeutically active groups per molecule, respectively. If one could "amplify," or increase, the number of such active groups per molecule, it would be possible to achieve enhancement of the intended medical result with less morbidity to the patient.
In my co-pending U.S. patent application, Ser. No. 06/928,943 ,now U.S. Pat. No. 4,863,717 of which the present application is a continuation-in-part, several novel techniques for providing long-lasting nitroxide-bearing MRI contrast agents were disclosed. The first technique involved the construction of liposomes, the lipid bilayers of which incorporated numerous long-chain nitroxides in the form of fatty esters. The nitroxides were exposed on the exterior of each liposome and an oxidant was encapsulated inside the liposome. Reduced nitroxides flipped to the inside of the liposome, became re-oxidized, then returned to the outside of the liposome. Because the nitroxides are thus "regenerated," the MRI contrast-enhancing effect imparted by a dose of such liposomes administered to a patient is long-lasting. Consequently, lower dosages are required to achieve a desired image contrast for a longer period of time than with previous nitroxide-based MRI contrast agents.
The second technique disclosed in my co-pending application involved the synthesis of molecules patterned after the radially symmetrical, branched molecules, termed arborols. The branches of the new molecules terminated with nitroxide groups. Such molecules can be administered to the patient in relatively low dosages for reduced toxicity and reduced osmotic disequilibria while still supplying high effective numbers of nitroxide groups for adequate MRI contrast enhancement during the time required for obtaining an MRI image.
There remains, however, a need for other diagnostic and therapeutic agents comprising molecules with multiple numbers of pharmacologically active groups, respectively, including agents that can be readily made bifunctional to become attachable either to specific biomolecules such as antibodies or to other loci within the body without the need to administer prohibitively large doses of the agents to the patient